RNA mutations in the sequence at the 5′ side of the terminator stem affect the rpmG-3′ and AniS-3′ binding to N. meningitidis ProQ protein. (A) Secondary structures of rpmG-3′ and its mutants. (B) The fitting of the ProQ binding data using the quadratic equation provided Kd values of 1.2 nM for rpmG-3′, 1.1 nM for rpmG-2AtoU, and 5.8 nM for rpmG-3AtoU. (C) Secondary structures of AniS-3′ and its mutants. (D) The fitting of the ProQ binding data using the quadratic equation provided Kd values of 4.9 nM for AniS-3′ and 2.6 nM for AniS-2UtoA. The fitting of AniS-3UtoA to the equation for one site-specific binding with the Hill slope model provided a Kd value of 0.3 nM. The data in the plots for rpmG-3′ and AniS-3′ binding to ProQ are the same as in Figure 1. The lower case g denotes guanosine residue added on the 5′ end to enable T7 RNA polymerase transcription. Green font indicates the introduced substitutions. Gels corresponding to the data in the plots are shown in Supplemental Figure S11. The RNA secondary structure predictions were performed in the ViennaRNA program (Lorenz et al. 2011). The average equilibrium dissociation constant (Kd) values and maximum RNA fraction bound calculated from at least three independent experiments are shown in Table 6.
